We have continued to improve the properties of our immunotoxins HA22 and HA22-LR directed at CD22. We have used alanine scanning mutagenisis to make an immunotoxin with improved affinity and activity. We have identified all the B cell epitopes in HA22-LR and made an immunotoxin with 8 mutations in domain III that retains all its cytotoxic activity, but does not induce antibody formation when injected 5 times i.v. into mice. We are now devising a method to determine the human B cell epitopes. We have also analyzed the residues in the furin sequence of HA22 and identified residues that make the immunotoxin more active. We have used information gained in the HA22 studies to improve the immunotoxin SS1P directed at mesotheliomas. We have introduced mutations into that immunotoxin to lower immunogenicity and made changes in the furin site that allows domain II of PE38 to be deleted without loss of cytotoxic activity. We have studied the mechanism of mesothelin shedding and tentatively identified the major enzyme involved in the shedding process. Inhibition of this enzyme could increase the activity of immunotoxins. In collaboration with D. FitzGerald we have begun to study how protein synthesis arrest leads to apoptosis and identified BAK and Mcl-1 as critical players in this process. In collaboration with Alan Wayne POB, we have analyzed the response of cells directly isolated from patients with ALL to HA22 killing and set up an assay that may help predict if patients will respond to treatment.